In the chemical and biotech field, target compounds such as drug candidates commonly need to be separated from contaminating species originating from the process of manufacture. For example, a protein drug candidate which has been produced by expression of recombinant host cells will need to be separated e.g. from the cells and possibly cell debris, other host cell proteins, DNA, RNA, and residues from the fermentation broth such as salts. Due to its versatility and sensitivity to the target compounds, chromatography is involved as at least one step in many of the currently used biotech purification schemes. The term chromatography embraces a family of closely related separation methods, which are all based on the principle that two mutually immiscible phases are brought into contact. More specifically, the target compound is introduced into a mobile phase, which is contacted with a stationary phase. The target compound will then undergo a series of interactions between the stationary and mobile phases as it is being carried through the system by the mobile phase. The interactions exploit differences in the physical or chemical properties of the components of the sample.
The stationary phase in chromatography is commonly comprised of a solid carrier to which ligands, which are functional groups capable of interaction with the target compound, have been coupled. Consequently, the ligands will impart to the carrier the ability to effect the separation, identification, and/or purification of molecules of interest. Liquid chromatography methods are commonly named after the interaction principle utilised to separate compounds. For example, ion exchange chromatography is based on ionic charge-charge interactions; hydrophobic interaction chromatography (HIC) utilises hydrophobic interactions; and affinity chromatography is based on specific biological affinities.
Thus, ion exchange is based on the reversible interaction between a charged target compound and an oppositely charged chromatography matrix. The elution is most commonly performed by increasing the salt concentration, but changes in pH are equally possible. Ion-exchangers are divided into cation-exchangers, wherein a negatively charged chromatography matrix is used to adsorb a positively charged target compound; and anion-exchangers, wherein a positively charged chromatography matrix is used to adsorb a negatively charged target compound. The term strong ion exchanger is used for an ion-exchanger which is charged over broad pH intervals, while a weak ion-exchanger is chargeable at certain pH values only. One commonly used strong cation-exchanger comprises sulphonate ligands (known as S groups). In some cases, such cation exchangers are named by the group formed by the functional group and its linker to the carrier; for example SP cation exchangers wherein the S groups are linked by propyl (P) to the carrier.
The properties of the carrier to which the ligands have been coupled will also affect the separation properties of the chromatography matrix. Depending on the intended mode of chromatography, carriers that are substantially hydrophilic or hydrophobic may be preferred. A further consideration of the carrier is the ease of which it is functionalized. Depending on the chemistry used for coupling ligands, the carrier may be activated i.e. transformed into a more reactive form. Such activation methods are well known in this field, such as allylation of the hydroxyl groups of a hydrophilic carrier such as dextran or agarose. Covalent ligand attachment is typically achieved using reactive functionalities on the solid support matrix such as hydroxyl, carboxyl, thiol, amino groups, and the like. In order to enhance the binding capacity of the matrix, a linking group known simply as a linker is often provided between the ligand and carrier. Such linkers will physically distance the ligand from the carrier thereby permitting the target compound to interact with the ligand with minimal interference from the matrix. However, the use of linkers in the synthesis of chromatography matrices requires the use of a functional reagent having at least one functional group capable of reacting with a functional group on the surface of the matrix to form a covalent bond therewith and at least one functional group capable of reacting with a functional group on the ligand to form a covalent bond therewith.
U.S. Pat. No. 5,789,578 (Massey University) relates to methods for the preparation of chromatography matrices comprising a support matrix having ligands capable of binding a target compound covalently attached thereto through a linking group comprising sulfide, sulfoxide, or sulfone functionality. Bisulphite is used as a reagent to provide S groups. More specifically, U.S. Pat. No. 5,789,578 uses allyl glycidyl ether, allyl halide or propargyl halide and conventional methods in the presence of a base to provide a carrier having ethylenically unsaturated entities pendent thereto. Specifically, the halide or glycidyl group reacts with matrix hydroxyl groups at alkaline pH. Under these conditions, the allyl group is expected to have limited reactivity with the matrix or water used in the reaction solution. The unsaturated group is then reacted under free radical conditions with bisulphite or a thiol-containing ligand to provide for covalent linkage thereof.
However, the introduction of allyl groups and subsequent coupling of S groups as disclosed in the above discussed U.S. Pat. No. 5,789,578 will leave a fraction of the allyl groups unreacted on the carrier while another fraction will be subject to the introduction of vicinal sulphonate-sulphinate groups. In addition, the activated reagents will also involve the risk of undesired cross-linking reactions. Further, it is known that the kind of reaction described in U.S. Pat. No. 5,789,578 will be initiated by oxygen, which oxygen is consumed in a competing reaction transforming the bisulphite to bisulphate. During the formation of sulphonate radicals, sulphate ions are also formed, which cause a drop in pH. To avoid deleterious effects of this pH drop, the reaction is commonly neutralized by addition of sodium hydroxide solution. The reaction is relatively time-consuming, and may be run e.g. overnight.
Thus, there is a clear need in this field of improved methods which allows functionalization of polysaccharide carriers in simpler ways avoiding the above- discussed disadvantages.